Enantiomeric separation of metoprolol and α-hydroxymetoprolol by liquid chromatography and fluorescence detection using a chiral stationary phase

A sensitive and stereoselective high-performance liquid chromatographic assay for the determination of the enantiomers of metoprolol (R- and S-) and the diastereoisomers of α-hydroxymetoprolol (IIA, IIB) in plasma is reported. Chromatography involved direct separation of enantiomers using a Chirobiotic T bonded phase column (250×4.6 mm) and a mobile phase consisting of acetonitrile–methanol–methylene chloride–glacial acetic acid–triethylamine (56:30:14:2:2, v/v). Solid-phase extraction using silica bonded with ethyl group (C2) was used to extract the compounds of interest from plasma and atenolol was used as the internal standard. The column effluent was monitored using fluorescence detection with excitation and emission wavelengths of 225 and 310 nm, respectively. S-Metoprolol, R-metoprolol, IIB and IIA eluted at about 5.9, 6.7, 7.3 and 8.2 min without any interfering peaks. The calibration curve was linear over the range of 0.5 to 100 ng/ml for each isomer of metoprolol and 1 to 100 ng/ml for each isomer of α-hydroxymetoprolol (IIA & IIB). The mean intra-run accuracies were in the range of 96.2 to 114% for R-metoprolol, 94.0 to 111% for S-metoprolol, 90.2 to 110% for IIA, and 94.6 to 106% for IIB. The mean intra-run precisions were all in the range of 2.2 to 12.0% for R-metoprolol, 2.1 to 11.1% for S-metoprolol, 1.9 to 14.5% for IIA, and 3.2 to 11.0% for IIB. The lowest level of quantitation for the enantiomers of metoprolol was 0.5 ng/ml and 1.0 ng/ml for α-hydroxymetoprolol (IIA and IIB). The absolute recoveries for each analyte was ≥95%. The validated method accurately quantitated the enantiomers of parent drug and metabolite after a single dose of an extended release metoprolol formulation.